CA2175042C - Method for production of substances using microorganisms with an increased productivity for nadph - Google Patents

Abstract

Productivity of target substances such as L-amino acids, antibiotics, vitamins, growth factors and physiologically active substances, which are produced by fermentation using a microorganism, is improved by increasing the productivity of reduced nicotinamide adenine dinucleotide phosphate in the cell of the microorganism.

Description

METHOD FOR PRODUCTION OF SUBSTANCES USING A MICROORGANISM
WITH AN INCREASED PRODUCTIVITY FOR NADPH
The present invention relates to the production of a target substance using a microorganism. More particularly, the invention is directed to a method of improving the productivity of a final target substance in the production of same using microorganisms.
Typical examples of the microorganism used in the present invention include microorganisms belonging to the genus Escherichia and coryneform bacteria, which have conventionally been used for production of various substances. Target substances produced herein include L-amino acids, antibiotics, vitamins, growth factors, physiologically active substances, and other substances generally produced by microorganisms.
L-amino acid fermentation is typically exemplified as <~ well known method for producing a target substance using microorganisms. L-amino acids are used not only for spices and foods but also as components of various medical nutrient mixtures, additives of animal feed, agents in pharmaceutical and chemical industries, and growth factors for the production of L-amino acids such as L-lysine and L-homoserine using microorganisms. Coryneform bacteria, microorganisms belonging to the genera Escherichia, Bacillus and SE:rratia and the like are known as microorganisms which can be used for the fermentation production of L-amino acids.
Wild type bacteria (wild type strains), auxotrophic strains induced from wild type strains, metabolic regulatory mutants induced from wild type strains as various drug resistant mutants, and strains having properties of both auxotrophic strains and metabolic regulatory mutants may be used for the fermentation production of L-amino acids. Substances required for the auxotrophic strains vary from strain to strain, and auxotrophic strains which require an identical substance vary in degree of those auxotrophy. Similarly, the metabolic regulatory mutants obtained as various drug resistant mutants have diversities.
Recombinant DNA technology has recently been used for L-amino acid fermentation. The theory of this technology is based on enhancement of an L-amino acid biosynthetic system in a host microorganism through enrichment of at least one gene coding for at least one L-amino acid biosynthetic enzyme. Details of such technology are described in, for example, Amino Acid Fermentation, Society Press, Japan (1986).
However, the microorganisms conventionally used in the fermentation production of L-amino acids have biosynthetic pathways including L-amino acid biosynthetic pathways and coenzyme biosynthetic pathways, identical with those of wild type microorganisms. The L-amino acid-producing microorganisms have been bred by desensitization of inhibition by a final product or the like existing in the L-amino acid biosynthetic pathway. To achieve such a breeding, for example, providing auxotrophic properties or drug resistance to the cell of a microorganism, or amplifying a gene coding for biosynthetic enzyme by recombinant DNA technology have been applied.
Many substances other than L-amino acids can be produced through fermentation using microorganisms.
Examples of such substances, include antibiotics and vitamins. There are various kinds of antibiotics and a variety of materials are used as precursors for the biosyntheses of such antibiotics. For example, sugars, amino acids, acetic acid, propionic acid and A

mevalonic acid are used. A target antibiotic is produced from such a precursor through a conversion process of various metabolites other than the precursor. The same mechanism is observed in vitamins and other biogenic substances.
In the production of the aforementioned substances using microorganisms, each substance is produced in a biosynthetic pathway in the cell of the microorganism. One of the important coenzymes essential for effective function of responsible enzymes in the biosynthetic system is reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as "NADPH"). However, the relationship between NADPH and the production of substances using microorganisms has not been reported.
Nicotinamide dinucleotide transhydrogenase (hereinafter referred to as "transhydrogenase") is known as one of the enzymes responsible for the production of NADPH. It is known that this enzyme is present in various organisms including microorganisms belonging to the genus Escherichia. In Escherichia coli, a typical microorganism belong to the genus Escherichia, purification of transhydrogenase (David M. Clarke and Philip D. Bragg, Eur. J. Biochem., 149, 517-523 (1985)), cloning of a gene coding for it (David M. Clarke and Philip D. Bragg, J.
Bacteriology, 162, 367-373 (1985)), and determination of a nucleotide sequence of the gene (David M.
Clarke, Tip W. Loo, Shirley Gillam, and Philip D.
Bragg, Euro. J. Biochem. 158, 647-653 (1986)) have been performed as well as making obvious the existence of the enzyme. However, a physiological function of the enzyme is still almost unknown. This A

is apparent from the fact that variants defective in the enzyme do not show any phenotypic expression.
It is an object of the present invention to improve the productivity of a target substance using a microorganism.
The productivity of a target substance has conventionally been improved by means of desensitization of regulation of synthesis by a final product or the like being produced by a biosynthetic pathway of the target substance and that of the coenzyme required for the synthesis of the target substance, which exists in the cells of the microorganisms. The object of the present invention is to provide a method of improving the productivity of a target substance according to a completely novel theory other than that mentioned above.
A variety of reducing reactions proceed in biosyntheses of substances such as L-amino acids in living organisms. In many cases, the coenzyme NADPH
is physiologically used as an intravital reducing substance. For example, glutamate dehydrogenase requires NADPH as a coenzyme in an L-glutamic acid biosynthetic pathway. Aspartate semialdehyde dehydrogenase, dihydrodipicolate reductase require NADPH as a coenzyme in an L-lysine biosynthetic pathway.
In other L-amino acid biosynthetic pathways, NADPH plays an important role as a coenzyme. In addition, L-glutamic acid is essential as a donor of amino groups in many L-amino acid biosynthetic pathways; therefore, NADPH is also required for the supply of amino groups in biosyntheses of L-amino acids.
NADPH is mostly prepared through metabolism of glucose in a pentose phosphate cycle in which A

glucose-6-phosphate dehydrogenase and phosphoglu-conate dehydrogenase are included. In the pentose phosphate cycle, the production efficiency of NADPH
can be calculated as being 12 molecules with respect to one molecule of glucose, since carbon dioxide is released.
On the other hand, reduced nicotinamide adenine dinucleotide (hereinafter referred to as "NADH") is a molecule extremely similar to NADPH; however, it cannot be utilized as a coenzyme for L-amino acid biosynthesis in most cases. NADH is biosynthesized through the TCA cycle, and a sufficient amount thereof is usually present in the cell.
As for L-amino acid biosynthesis pathways, intravital components which cannot be effectively utilized are often produced through the process of biosynthesis of desired L-amino acids from glucose.
Such components are ordinarily oxidized through the TCA cycle, resulting in the generation of a large amount of NADH.
Applicant has established a first hypothesis that a large amount of NADPH is required during production of a target substance using a microorganism, that glucose is inevitably consumed to supply such NADPH and, consequently, the productivity of the target substance per consumed sugar is lowered.
Further, Applicant has established a second hypothesis that intravital components which cannot be effectively utilized during the production of a target substance are inevitably accumulated in the production process of the target substance using a microorganism, and that these are metabolized through the TCA cycle, resulting in an increase of the NADH
concentration in the cell.
A

It is assumed on the basis of the above described first and second hypotheses that if intracellular NADH
can be efficiently converted into NADPH, sugar required for biosynthesis of NADPH by a microorganism can be saved, and a target substance can be produced at a higher productivity. It is also assumed that transhydrogenase may be utilized for converting NADH generated through the TCA cycle into NADPH.
As a result of vigorous studies, Applicant has succeeded in obtaining a DNA fragment containing a transhydrogenase gene from a bacterium belonging to the genus Escherichia, and in showing that the abilities of microorganisms to produce reduced nicotinamide adenine dinucleotide phosphate are enhanced by using the DNA
fragment. Applicant has further found that the productivity of the target substance is improved in the above-mentioned microorganisms having enhanced abilities to produce reduced nicotinamide adenine dinucleotide phosphate.
In accordance with the present invention, there is thus provided a method for the production of a target substance using a microorganism, comprising the steps of:
a) Cultivating a microorganism in a culture medium to produce the target substance and accumulate same in said culture medium, said microorganism having been modified so that productivity of said microorganism for reduced nicotinamide adenine dinucleotide phosphate from reduced nicotinamide adenine dinucleotide is increased;
b) Collecting the target substance from said culture medium.
Preferably, the target substance is an L-amino acid. Examples of L-amino acids which may be produced according to the invention include L-threonine, L-lysine, L-glutamic acid, L-leucine, L-isoleucine, L-valine and L-phenylalanine.
The microorganism preferably used is a microorganism belonging to the genus Escherichia or a coryneform bacterium.
In a preferred embodiment of the invention, step (b) is carried out by increasing an enzyme activity of nicotinamide nucleotide transhydrogenase in the cell of the microorganism.
According to another preferred embodiment, step (b) is carried out by increasing an expression amount of a gene coding for nicotinamide nucleotide transhydrogenase in the cell of the microorganism.
According to a further preferred embodiment, step (b) is carried out by increasing a number of copies of a gene coding for nicotinamide nucleotide transhydrogenase in the cell of the microorganism.
Other than L-amino acids, any target substance may be produced provided that NADPH is required for its biosynthesis, including those which have been hitherto produced by microorganisms, for example, nucleic acid such as guanylic acid and inosinic acid, vitamins, antibiotics, growth factors, and physiologically active substances. Even in the case of a substance which has not been produced using a microorganism until now, the method of the present invention can be applied provided that NADPH is required for its biosynthesis.
In the biosynthesis of streptomycin, for example, NADPH is used for synthesis of dTDP-4-oxo 4,6-dideoxy-D-glucose from dTDP-glucose. In addition, amino acids serve as precursors in the case of peptide antibiotics, and thus NADPH is required for their biosynthesis. Further, precursors of penicillin, a beta-lactam antibiotic, are L-valine, A

- $ - 2175042 L-cysteine and L-alpha-aminoadipic acid, and thus NADPH is required for their biosynthesis.
When it is intended to know whether a substance requires NADPH in its biosynthesis, the fact may become apparent from a biosynthetic pathway if its biosynthetic pathway has been revealed.
The microorganism to be used in the present invention is not especially limited, provided that it belongs to those which have been hitherto used for the production of substances, such as bacteria belonging to the genus Escherichia, coryneform bacteria, bacteria belonging to the genus Bacillus, and bacteria belonging to the genus Serratia. It is preferably a microorganism in which a DNA fragment containing a replication origin of a plasmid has been obtained for the microorganism, a transhydrogenase gene function, and a number of copies of the transhydrogenase gene can be increased. On the other hand, a strain which originally has a high ability to produce a target substance is most preferred for improving productivity according to the present invention. For example, use can be made of Escherichia coli B-3996 strain and the like when the target substance is L-threonine, Escherichia coli AJ12604 (FERM BP-3579) strain and the like when the target substance is L-phenylalanine, Escherichia coli AJ12624 (FERM BP-3853) strain and the like when the target substance is L-glutamic acid, and Brevibacterium lactofermentum AJ3990 (FERM P-3387, ATCC 31269) and the like when the target substance is L-lysine.
No problem occurs in respect of the medium to be used for producing the target substance when a well-known medium having been hitherto employed is used depending on the microorganism utilized. For example, A

an ordinary medium which contains a carbon source, a nitrogen source, inorganic ions and optionally other organic components can be used. No special medium is required for carrying out the present invention.
As the carbon source, it is possible to use sugars such as glucose, lactose, galactose, fructose and starch hydrolysate; alcohols such as glycerol and sorbitol; and organic acids such as fumaric acid, citric acid and succinic acid or the like.
As the nitrogen source, it is possible to use inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate; organic nitrogen such as soy bean hydrolysate; ammonia gash and aqueous ammonia.
As for organic trace nutrient sources, use can be made of substances such as vitamin Bl, L
homoserine and L-tyrosine or yeast extract. Other than the above, small amounts of potassium phosphate, magnesium sulfate, iron ion, manganese ion and the like are optionally added.
Cultivation may be performed under well-known conditions which have been hitherto employed and which depends on the microorganism utilized. It is preferable to perform the cultivation for 16-120 hours under an aerobic condition. The cultivation temperature is controlled to 25-45°C, and pH is controlled to 5-8 during cultivation. Inorganic or organic acidic or alkaline substances as well as ammonia gas and the like may be used for pH
3 0 adj ustment .
No special method is required for collecting the metabolic product from the culture medium after completion of the cultivation. For example, well known methods such as an ion-exchange resin method, a precipitation method and the like can be employed.
A

- 1~ - 2175042 The NADPH productivity of the microorganism is preferably increased by increasing the enzyme activity of transhydrogenase in the microbial cells.
The enzyme activity of transhydrogenase can be increased by increasing an expression amount of a transhydrogenase gene in the mocrobial cells. It is also possible to increase the enzyme activity of transhydrogenase by modifying a transhydrogenase gene and creating a transhydrogenase with increased activity.
The expression amount of a transhydrogenase gene in the microbial cells is preferably increased by increasing a number of copies of the transhydrogenase gene in the microbial cells.
In order to increase the number of copies of the transhydrogenase gene, a DNA fragment containing the aforementioned gene is necessary. Incidentally, a transhydrogenase gene has been cloned in an Escherichia coli K-12 strain as a bacterium belonging to the genus Escherichia, and its nucleotide sequence has been determined (D. M. Clarke et al., Eur. J.
Biochem., 158, 647-653 (1986)). Thus, preparation of a DNA fragment containing the aforementioned gene is achieved by using the method disclosed by D. M. Clarke et al. Further, a desired DNA fragment can be obtained by a hybridization method using a synthetic DNA probe prepared with reference to a nucleotide sequence disclosed by D. M. Clarke et al., and a PCR method using synthetic DNA primers prepared with reference to the aforementioned nucleotide sequence. If the DNA fragment containing the transhydrogenase gene is ligated with a vector DNA
autonomously replicable in a target microorganism and inserted into the aforementioned microorganism, it is A

possible to increase the number of copies of the transhydrogenase gene.
The DNA primer, which is used upon cloning of the transhydrogenase gene from a bacterium belonging to the genus Escherichia by using the PCR method, may be appropriately prepared on the basis of, for example, the sequence known in Escherichia coli (D.
M. Clarke et al., Eur. J. Biochem., 158, 647-653 (1986)). Since transhydrogenase comprises two subunits, it may be necessary to amplify both pntA
and pntB genes of each of them. Two primers of 5'-CTGATTTTTGGATCCAGATCACAG-3' (SEQ ID N0:1) and 5'-CGTTCTGTTAAGCTTTCTCAATAA-3' (SEQ ID N0:2), which can amplify a region of 3 kb containing both pntA and pntB genes, are suitable. These primers are slightly different from the sequence report by D. M. Clarke et al. However, owing to the change in sequence, it is possible to introduce a BamHI cleavage site upstream from both the pntA and pntB genes, and a HindIII
cleavage site downstream from both the pntA and pntB
genes. Neither the BamHI site nor the HindIII site exists in both the genes and in the vicinity thereof.
Thus, they are convenient upon cloning of am amplified DNA fragment by using these restriction enzymes, and upon transfer into another vector DNA.
Synthesis of the primer DNA can be performed in accordance with an ordinary method by using a DNA
synthesizer Model 380B produced by Applied Biosystems, and by using the phosphoamidite method (see Tetrahedron Letters, 22, 1859 (1981)). The PCR
method can be performed by using a DNA Thermal Cycler Model PJ2000 produced by Takara Shuzo Co., Ltd., and by using Tag DNA polymerise in accordance with the method designated by the manufacturer.
A

The DNA fragment containing the transhydrogenase gene can be obtained from microorganisms other than the bacteria belonging to the genus Escherichia. A
desired DNA fragment can be obtained by using a hybridization method using a synthetic DNA probe prepared with reference to the nucleotide sequence disclosed by D. M. Clarke et al . , or by using a PCR
method using synthetic DNA primers prepared with reference to the aforementioned nucleotide sequence, as the method for obtaining it.
The DNA probe to be used for the hybridization method or the DNA primers to be used upon cloning of the gene using the PCR method may be appropriately prepared on the basis of, for example, the sequence known in Escherichia coli (D. M. Clarke et al., Eur.
J. Biochem., 158, 647-653 (1986)). It is postulated that the nucleotide sequence of the gene is different for each of the microorganisms. Thus, it is desirable to prepare synthetic DNAs matching with portions conserved with respect to transhydrogenases originating from each of the microorganisms.
The transhydrogenase gene amplified by the PCR
method is ligated with a vector DNA autonomously replicable in a cell of a bacterium belonging to the genus Escherichia when it is introduced into the bacterium belonging to the genus Escherichia, and introduced into the cells of the bacterium belonging to the genus Escherichia.
In the case of introduction of the obtained DNA
fragment containing a transhydrogenase gene into a microorganism other than the bacteria belonging to the genus Escherichia, the aforementioned DNA
fragment is ligated with a vector DNA autonomously replicable in a cell of the microorganism subjected to the introduction of the aforementioned DNA
A

fragment, and introduced into the aforementioned cells.
A plasmid vector DNA is preferable as the vector DNA capable of being used in the present invention for example, use can be made of pUCl9, pUCl8, pBR322, pHSG299, pHSG399, RSF1010 and the like. Other than the above, phage DNA vectors are also available. In order to efficiently achieve expression of transhydrogenase, it is also acceptable to use a promoter workable in microorganisms such as lac, trp and PL. In addition, in order to increase the number of copies of the transhydrogenase gene, DNA
containing the aforementioned gene may be integrated into a chromosome by means of a method using transposon (Berg D. E. and Berg, C. M., Bio/Technol., 1, 417 (1983)), Mu phage (Japanese Patent Laid-open No. 2-109985), or homologous recombination (Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)).
When the microorganism into which the gene is introduced is a coryneform bacterium, the vector DNA
which can be used in the present invention is a plasmid vector autonomously replicable in the coryneform bacterium, such as pAM330 (see Japanese Patent Publication No. 1-11280) or pHM1519 (see Japanese Patent Laid-open No. 58-77895).
In order to select a strain having actually enhanced enzyme activity of transhydrogenase among candidate strains having potentially enhanced enzyme activity of transhydrogenase, for example, a known method (David M. Clarke and Philip D. Bragg, Journal of Bacteriology, 162, 367-373 (1985)) may be used as a method for confirming the enhancement of enzyme activity of transhydrogenase.
In the accompanying drawings:
A

Fig. 1 schematically illustrates the preparation of plasmid pMW::THY;
Fig. 2 schematically illustrates the preparation of plasmid pHSG::THYB; and Fig. 3 schematically illustrates the preparation of plasmid pSU::THY.
The following non-limiting examples illustrate the invention.
EXAMPLE 1: Cloning of Transhydrogenase Gene Nucleotide sequences of the genes pntA and pntB
which code for transhydrogenase of Escherichia coli, have been determined previously (D. M. Clarke et al., Euro. J. Biochem. 158, 647-653 (1986)), and it has been reported that pntA and pntB code proteins of 502 amino acid residues and of 462 amino acid residues, respectively. In addition, it is also known that both these proteins are required for expression of an enzymatic activity of transhydrogenase, that both the pntA and pntB genes are located in series on a chromosomal DNA, and that both genes can thereby be cloned in one DNA fragment.
For the convenience of subsequent operations, Applicant has simultaneously cloned not only both the pntA and pntB genes but a region existing in the upstream of these genes, which has a promoter activity. Thus, a DNA fragment containing the genes and the promoter region were amplified by the PCR
method to clone it.
Two synthetic oligonucleotides having the sequence of 5'-CTGATTTTTGGATCCAGATCACAG-3' (SEQ ID
N0:1) and 5'-CGTTCTGTTAAGCTTTCTCAATAA-3' (SEQ ID
N0:2) were synthesized as primers for the PCR method.
As a template DNA for the PCR method, total genome DNA of Escherichia coli K-12 MC1061 was prepared according to the method of Saitoh and Miura (Biochem.
i~,;~

Biophys. Acta., 72, 619, (1963)). A target DNA
fragment was amplified by the PCR method using the two primer oligonucleotides and template chromosomal DNA according to the method of Erlich et al. (PCR
Technology, Stockton press (1989)). The synthetic DNAs used as primers have nucleotide sequences with little difference in the respective central portions of the synthetic DNA fragments from the nucleotide sequence reported by D. M. Clarke et al. This is schemed to introduction of the BamHI cleavage site and the HindIII cleavage site in design of the synthetic oligonucleotides. These restriction enzyme cleavage sites are required for insertion of the amplified DNA fragment into the vector DNA.
Introduction of the restriction sites causes a mismatch between the primers and the chromosomal DNA
in the process of PCR. However, the mismatch did not affect the amplification of the DNA by the PCR
method, since these restriction sites were located in the central portions of the synthetic DNA fragment.
Amplification of the DNA fragment of 3.0 kb was confirmed by agarose gel electrophoresis.
The amplified 3.0 kb of DNA fragment and a plasmid vector pMW118 having an ampicillin resistant marker (available from Nippon Gene Inc.) were digested with BamHI and HindIII. Both the digested DNA fragment and a vector DNA were ligated to produce a recombinant DNA with DNA ligase. The resulting recombinant plasmid was named as pMW::THY (see Fig.
1) .
Escherichia coli JM109 (available from Takara Shuzo Co., Ltd.) was transformed with the plasmid pMW::THY and the transformant Escherichia coli JM109 (pMW::THY) was obtained. The enzyme activity of transhydrogenase existing in each of the cell extract A

solutions of Escherichia coli JM109 and Escherichia coli JM109 (pMW::THY) was measured according to a known method (David M. Clarke and Philip D. Bragg, J.
Bacteriology, 162, 367-373 (1985)). The results are shown in Table 1.

Strains JM109 JM109 (pMW::THY) Transhydrogenase Specific Activity 1.0 1.7 (ug/mg protein) As shown in Table l, it is apparent that Escherichia coli JM109 (pMW::THY) has a higher enzyme activity of transhydrogenase than Escherichia coli JM109. This proves that the DNA fragment inserted into the plasmid pMW::THY includes the transhydrogenase gene. Escherichia coli harboring the plasmid pMW::THY was designated as strain AJ12929.
The strain AJ12929 has been deposited in the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, Japan on October 4, 1993, under deposition number FERM P-13890, and was transferred from the original depository to an international depository based on the Budapest Treaty on September 14, 1994, where it was deposited under the deposition number FERM BP-4798.
The plasmid DNA of pMW::THY was prepared according to a conventional method and digested with BamHI and HindIII to isolate a 3.0 kb DNA fragment containing the transhydrogenase gene. The plasmid vector pHSG399 having a chloramphenicol resistant A

- 1' - 2175042 marker (available from Takara Shuzo Co., Ltd.) was cleaved with BamHI and HindIII and the large fragment was isolated. Then the DNA fragment containing the transhydrogenase gene and the BamHI-HindIII large fragment of pHSG399 were ligated with DNA ligase to obtain plasmid pHSG::THY.
The plasmid pHSG::THY can autonomously replicate in the cells of microorganisms belonging to Escherichia, but is not stable in the cells of coryneform bacteria. Thus, a replication origin obtained from an autonomous replicable plasmid derived from coryneform bacterium was introduced into the plasmid pHSG::THY.
A plasmid pHM1519 autonomously replicable in the cells of coryneform bacteria (see Japanese Patent Laid-open No. 58-77895) was cleaved with a restriction enzyme BamHI to obtain a DNA fragment of 3.0 kb containing a replication origin. On the other hand, the pHSG::THY plasmid was cleaved with BamHI to obtain a DNA fragment. Both DNA fragments were ligated with DNA ligase to produce plasmid pHSG::THYB
(Fig. 2). A strain of Escherichia coli harboring pHSG::THYB was designated as AJ12872 strain. This AJ12872 strain has been deposited in the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, the Ministry of international Trade and Industry, Japan on October 4, 1993 under the deposition number FERM P-13889 and was transferred from the original depository to an International depository based on the Budapest Treaty on September 14, 1994, where it was deposited under deposition number FERM BP-4797.
Further, a plasmid pSUl8 which is autonomously replicable in the cells of bacteria belonging to the genus Escherichia and has a kanamycin resistant ,,, marker (Borja, Bartolome et al., Gene, 102, 75-78 (1991) was cleaved with restriction enzymes BamHI and HindIII to obtain a large fragment. This large fragment was ligated with the DNA fragment of 3.0 kb containing the transhydrogenase gene described above by using DNA ligase to produce plasmid pSU::THY (Fig.
3). An Escherichia coli strain harboring pSU::THY was designated as AJ12930 strain. This AJ12930 strain has been deposited in the National Institute of Bioscience and Human Technology, Agency of Industrial Microorganism Breeding, Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, Japan on October 4, 1993 under the deposition number FERM P-13891 and was transferred from the original depository to an international depository based on the Budapest Treaty on September 14, 1994, where it was deposited under deposition number FERM BP-4799.
The above confirmed that the productivity of various L-amino acids in the cells of the bacterium belonging to the genus Escherichia or the coryneform bacterium was affected by increasing the transhydrogenase activity in both bacteria.
EXAMPLE 2: Fermentation Production of L-Threonine by Strain with Introduced Transhydrogenase As L-threonine-producing bacteria of Escherichia coli, a B-3996 strain (see Japanese Patent Laid-open No. 3-501682 has the highest production ability among those known at present. Thus, the B-3996 strain was used as a host for evaluating the effect of enhancement of transhydrogenase. The B-3996 strain harbors a plasmid pZTIC40 (International Publication No. W090/04636) which was obtained by insertion of threonine operon in a broad-host range vector plasmid pAYC32 having a streptomycin resistant marker (cf.

Chistoserdov, A. Y. and Tsygenkov, Y. D., Plasmid, 1986, _16, 161-167). The B-3996 strain has been deposited in the Research Institute for Genetics and Industrial Microorganism Breeding under the deposition number RIA1867.
The PMW::THY plasmid was recovered from the Escherichia coli AJ12929 obtained in Example 1 by the method of Maniatis et al. (Sambrook, J., Fritsch, E.
F., Maniatis, T., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1, 21 (1989)). The pMW::THY
plasmid thus obtained was introduced into the B-3996 strain by the method of D. M. Morrison (Methods in Enzymology, 68, 326, (1979)). The B-3996 strain transformed with pMH::THY was designated as B-3996(pMW::THY) strain. The B-3996 strain and the B-3996(pMW::THY) strain were cultivated under the following conditions.
Cultivation was performed for 38 hours at a temperature of 37°C with a stirring at 114-116 rpm using a medium having the composition shown in Table 2. In Table 2, components A, B and C were separately prepared, sterilized and cooled, and mixed using a 16/20 volume of component A, a 4/20 volume of component B, and 30 g/1 of component C. The results of the cultivation are shown in Table 3. As it is apparent, the L-threonine productivity was improved by enhancing the intracellular transhydrogenase activity in an L-threonine-producing bacterium belonging to the genus Escherichia.
A

(A) (NH4)2504 16 g/1 KH2P04 1 g/1 MgS047H20 1 g/1 FeS047H20 0.01 g/1 MnS045H20 0.01 g/1 Yeast Ext. (Difco) 2 g/1 L-Met 0.5 g/1 Adjusted to pH 7.0 with KOH and autoclaved at 115°C for 10 minutes 16/20 volume) (B) 20o Glucose Autoclaved at 115°C for 10 minutes (4/20 volume) (C) Pharmacopoeia CaC03 Autoclaved at 180°C for 2 days (30 g/1) Antibiotics (streptomycin: 100 ~g/ml, kanamycin:
5 ~g/ml ) Strains B-3996 B-3996(pMW::THY) Threonine 12.97 13.99 (g/1) EXAMPLE 3: Fermentation Production of L-Lysine by Strain with Introduced Transhydrogenase This experiment was carried out with a view to confirming whether or not the L-lysine productivity was improved by enhancing intracellular transhydrogenase activity in an L-lysine-producing coryneform bacterium. As L-lysine-producing bacterium belonging to the coryneform bacteria, a Brevibacterium lactofermentum AJ3990 strain was used.
A

The AJ3990 strain has been deposited in the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, Japan, under the deposition number FERM P-3387. The pHSG::THYB plasmid was recovered from the Escherichia coli AJ12872 strain obtained in Example 1 by the method of Maniatis et al. (Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1, 21 (1989)). The plasmid thus obtained was introduced into the AJ3390 strain by means of a transformation method using electric pulse (see Japanese Patent Laid-open No. 2-207791). The AJ3990 strain transformed with pHSG::THYB was designated as the AJ3390(pHSG::THYB) strain. The AJ3990 strain and the AJ3390(pHSG::THYB) strain were cultivated under the following conditions.
Cultivation was performed for 72 hours at a temperature of 31.5°C with stirring at 114-116 rpm using a medium having the composition shown in Table 4. Three different sugars, glucose, sucrose and fructose were used as sugar. The results of the cultivation are shown in Table 5. As it is apparent, the L-lysine productivity was improved by enhancing the intracellular transhydrogenase activity in an L-lysine-producing coryneform bacterium.

Sugar 36 g/1 NH4C1 20 g/1 KH2P04 1 g/1 MgS047H20 0.4 g/1 FeS047H20 10 mg MnS044H20 8 mg A

Soy bean protein hydrolysate (as nitrogen) 1 mg Thiamine-HC1 100 mg Biotin 300 mg After autoclaving at 115°C for 10 minutes, 3~ calcium carbonate separately sterilized was added.

Lysine hydrochloride accumulation amount (g/1) AJ3990 AJ3990(pHSG::THYB) Glucose 13.6 14.5 Sucrose 11.1 12.6 Fructose 8.2 11.9 EXAMPLE 4: Fermentation Production of L-Phenylalanine by Strain with Introduced Transhydrogenase This experiment was carried out with a view to confirming whether or not L-phenylalanine productivity was improved by enhancing intracellular transhydrogenase activity in an L-phenylalanine-producing bacterium belonging to the genus Escherichia. As the L-phenylalanine-producing bacterium belonging to the genus Escherichia, an Escherichia coli AJ12604 strain was used. AJ12604 strain harbors a plasmid pBR-aroG4 which was obtained by insertion of mutant aroG gene in a vector plasmid pBR322 having an ampicillin resistant marker and a plasmid pACMAB which was obtained by insertion of mutant pheA gene in a vector plasmid pACYC184 having a chloramphenicol resistant marker (see Japanese Patent Laid-open No. 5-236947). The AJ12604 strain has been deposited in the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, Japan on January 28, 1991 under the deposition number FERM P-11975, and was transferred from the original depository to an international depository based on the Budapest Treaty on September 26, 1991, where it was deposited under the deposition number FERM BP-3579. The pSU::THY
plasmid was recovered from the Escherichia coli AJ12930 strain obtained in Example 1 by the method of Maniatis et al. (Sambrook, J., Fritsch, E.F., Maniatis, T., Molecular Cloning, Cold Spring Harbor Laboratory Press, l, 21 (1989)). The plasmid thus obtained was introduced into the AJ12604 strain by the method of D. M. Morrison (Methods in Enzymology, 68, 326 (1979)). The AJ12604 strain transformed with pSU::THY was designated as AJ12604(pSU::THY) strain.
The AJ12604 strain and the AJ12604(pSU::THY) strain were cultivated under the following conditions.
Cultivation was performed for 16 hours at a temperature of 37°C with stirring at 114-116 rpm using a medium having the composition shown in Table 6. The results of the cultivation are shown in Table 7. As it is apparent, the L-phenylalanine productivity was improved by enhancing the intracellular transhydrogenase activity in an L-phenylalanine-producing bacterium belonging to the genus Escherichia.
T:
M.

Glucose 20 g/1 Na2HP04 29.4 g/1 KH2P04 6 g/1 NaCl 1 g/1 NH4C1 2 g/1 Sodium citrate 20 g/1 Sodium L-glutamate 0.4 g/1 MgS047H20 3 g/1 CaCl2 0.23 g/1 Thiamine-HC1 2 mg L-Tyrosine 75 mg After autoclaving at 115C for 10 minutes, 3% calcium carbonate separately sterilized was added.

Strain AJ12604 AJ12604(pSU::THY) 1-phenylalanine 4.28 4.89 accumulation 3.75 4.28 amount g/1 A

SEQENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Ajin~noto Co. Inc.
(ii) TITLE OF INVENTION: Method for Production of Substances (iii) NUMBER OF SEQUENCES:2 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE:
(B) STREET:
(C) CITY:
(D) STATE:
(E) COUNTRY:
(F) ZIP:
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME:
(B) REGISTRATION NUMBER:
( C ) REFERErICE/DOC~CET NUMBER:
(ix) TELECONW(JNICATION INFORMATION:
(A) TELEPHONE:
(B) TELEFAX:
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 (H) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other..synthetic DNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 (B) TYPE: nucleic acid v (C) STRANDEDNESS: single ' (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: other..synthetic DNA

1~~a4~.
_ _ ~_ (iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

Claims (9)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for the production of a target substance using a microorganism, comprising the steps of:
a) Cultivating a microorganism in a culture medium to produce the target substance and accumulate same in said culture medium, said microorganism having been modified so than productivity of said microorganism for reduced nicotinamide adenine dinucleotide phosphate from reduced nicotinamide adenine dinucleotide is increased by increasing an enzyme activity of nicotinamide nucleotide transhydrogenase in the cell of said microorganism;
b) Collecting the target substance from said culture medium.

2. A method according to claim 1, wherein said target substance is an L-amino acid.

3. A method according to claim 2, wherein said L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-glutamic acid, L-leucine, L-isoleucine, L-valine and L-phenylalanine.

4. A method according to claim 1, wherein said microorganism is a microorganism belonging to the genus Escherichia.

5. A method according to claim 2, wherein said microorganism is a microorganism belonging to the genus Escherichia.

6. A method according to claim 1, wherein said microorganism is a coryneform bacterium.

7. A method according to claim 2, wherein said microorganism is a coryneform bacterium.

8. A method according to claim 1, wherein said enzyme activity is increased by increasing an expression amount of a gene coding for nicotinamide nucleotide transhydrogenase in the cell of said microorganism.

9. A method according to claim 8, wherein said expression amount is increased by increasing a number of copies of a gene coding for nicotinamide nucleotide transhydrogenase in the cell of said microorganism.